Project summary/abstract Alterations in the excitable domains of myelinated axons, specifically the axon initial segment (AIS) and the nodes of Ranvier, are key pathophysiologies in various neurodegenerative conditions, including diabetes. Shortening of AIS length has been shown to lower neuronal excitability, and is also implicated in cognitive impairment in type 2 diabetes and Alzheimer?s disease. However, the cellular and molecular mechanisms of how these domains are altered in disease conditions remain poorly understood. This critical gap in knowledge limits the field?s ability to manipulate the AIS and nodes for treatment. The current proposal seeks to elucidate this important aspect of nervous system pathophysiology. The overall objective of this application is to identify a critical molecular link in the process of AIS and nodal disruption. The prior studies and preliminary data provided here have identified elevations in methylglyoxal (MG), a highly reactive byproduct of glucose metabolism, as a potential mediator for AIS and nodal disruption. These data also support that calpains, calcium-dependent intracellular cysteine proteases, are involved in this process. The central hypothesis is that methylglyoxal disrupts AIS and nodal protein complexes via calpain activation and inhibits nervous system function. We will test this hypothesis via three Specific Aims. Aim 1: Test the hypothesis that reduction of MG levels with novel scavenging peptides will ameliorate AIS shortening and cognitive impairment in db/db mice, an established model for type 2 diabetes. Aim 2: Test the hypothesis that elevated MG causes AIS/node changes, reduced neural network activity (Aim 2A, in vitro; mouse cortical neuron culture and multi-electrode arrays), and cognitive impairment (Aim 2B, in vivo; systemic administration of MG or inhibitor of glyoxalase 1, an enzyme that detoxifies MG, in wild-type mice). Aim 3: Test the hypothesis that calpains mediate the effects of MG on AIS/node structures, neural network activity (Aim 3A, in vitro; pharmacological calpain inhibition), and cognitive function (Aim 3B, C, in vivo; genetic manipulation of calpastatin, a specific endogenous inhibitor of calpains). Aim 3B will assess combined effects of increased MG and calpain over-activation in calpastatin knockout mice; and Aim 3C will assess increased MG and calpain inhibition in mice over-expressing calpastatin. This application is conceptually innovative, as we propose that the key targets of elevated MG are the structures of the AIS and nodes of Ranvier in live neurons. Innovative use of multi-electrode arrays will determine the effects of increased MG and AIS shortening on neural network function. The proposed research is significant, because completion of the aims will validate MG and calpains as potential targets for translational research aimed at treatments ? such as the novel MG scavengers tested in Aim 1 ? for comorbid cognitive impairment in type 2 diabetes. These results also have potential to impact a wide variety of neurodegenerative conditions, such as Alzheimer?s, thus ultimately providing a sustained and powerful influence on the field.